Because of high performance of polymerase chain reaction (PCR), its applied fields have been expanded year by year (Randall K,. Saiki et al. (1988) Science 239, 487-491). Even a DNA fragment of one molecule can be amplified by the PCR. A direct sequencing method, which is one for sequencing a product amplified by the PCR, is also useful (Corinne Wong et al. (1988) Nature, 330, 384-386). This method enables to reveal information concerning sequence of numerous samples quickly and simultaneously without preparation of libraries and screening thereof.
The direct sequencing method, however, suffers from two serious problems.
First, primers and 2'-deoxyribonucleoside-5'-triphosphates (2'-dNTPs) which have not incorporated remain in reaction systems and interfere the sequencing reaction. Accordingly, the residual primers and 2'-dNTPs must be removed from the PCR products before sequencing in conventional methods. Various kinds of methods for purification of the PCR products are known and which include purification by electrophoresis, ethanol precipitation, filtration, HPLC and the like (see, for example, Dorit R. L. et al. (1991) Current Protocols in Molecular Biology, Vol .II, John Wiley and Sons, New York, 15.2.1-15.2.11). However, all of these methods are complicated.
Second problem is the quick renaturation of PCR products. When the PCR products are renatured into a double-stranded DNA, they are no longer a single-stranded template and interfere the annealing between primers and single-stranded templates. For minimizing the renaturation, there have been proposed, for example, quenching after the denaturation, biotilation of one primer and adsorption of PCR products onto a streptavidin coated surface, use of exonuclease, asymmetric PCR and the like (see, for example, Barbara Bachmann et al. (1990) Nucleic Acid Res. Vol. 18, 1309). However, most of these methods are time-consuming and complicated.
Therefore, an object of the present invention is to provide a completely novel method for determining DNA nucleotide sequence, which does not require to remove the primers and 2'-deoxyribonucleoside-5'-triphosphates (2'-dNTPs) unreacted and remained in PCR reaction systems and which also does not require denaturation so that the problem of quick renaturation of PCR products could be obviated.